Project summary G protein-coupled receptors (GPCRs) comprise the most abundant human membrane protein family, representing 4% of the protein-coding genome. Due to their numerous physiological and pathological roles, approximately 34% of marketed drugs target a GPCR. Although GPCRs are relevant druggable targets ~140 are still orphans i.e. their endogenous ligands unidentified. The identification of the endogenous molecules that activate the receptor is an essential step that helps define the receptor?s role in biological pathways. Here we describe studies to screen orphan GPCRs using genome editing and a high-throughput screening platform, testing each receptor against 48 distinct peptides that are predicted to be neuropeptides based on their abundance in brain, their localization to the regulatory secretory pathway, and sequence conservation. We have carefully selected 18 GPCRs that are potential neuropeptide-binding receptors based on their distribution, expression levels, and sequence conservation. We will use two complementary approaches for deorphanization: in Aim 1 we will use a loss-of-function strategy by generating GPCR knockout cell lines using CRISPR/Cas9 system; and in Aim 2 we will use a gain-of-function strategy by generating stable GPCR overexpression cell lines. A high-throughput screening assay will be used to measure the peptide ligand-induced GPCR activation- mediated changes in the intracellular calcium release in these two cellular models. Using these two independent approaches (loss- and gain- of-function) we have previously successfully deorphanized two different orphan GPCRs and hence we are confident that the proposed studies will be successfully carried out within the one- year period of this award. The results obtained from this proposal will guide future studies that explore physiological roles of the receptors as well as facilitate structural studies that seek insights into receptor-ligand binding sites, which are fundamental to the development of new therapeutics targeting the receptors.